CN113933423A - Detection method for measuring 23 psychotropic drugs and metabolites in human serum - Google Patents
Detection method for measuring 23 psychotropic drugs and metabolites in human serum Download PDFInfo
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Abstract
The invention provides a detection method for detecting 23 psychotropic drugs and metabolites in human serum, which belongs to the technical field of psychotropic drug and metabolite detection, wherein 9 stable isotope internal standards are mainly used, a pair of quantitative ion pairs is respectively selected for each substance to be detected, the corresponding retention time of the quantitative ion pairs is taken as a qualitative basis, a standard curve is made for quantification by using a standard product, and the accuracy and the effectiveness of a low-level, medium-level and high-level quality control product inspection method are applied, so that the matrix effect can be effectively avoided; the detection method provided by the invention has the characteristics of simple and convenient sample pretreatment, small required sample volume, high sensitivity, high accuracy and high flux.
Description
Technical Field
The invention belongs to the technical field of detection of psychotropic drugs and metabolites, and particularly relates to a method for simultaneously and quantitatively analyzing the concentrations of 23 psychotropic drugs and metabolites in human serum based on an LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry) technology.
Background
In the psychiatric department, there are approximately 200 drugs that have been developed successively in the past 60 years and are available, which have become effective and indispensable drugs for the treatment of psychiatric disorders and symptoms. Therapeutic Drug Monitoring (TDM) is a very effective method for tailoring the dosing regimen to different individual patients. The main reason for using TDM to guide psychotropic medication is the significant individual variability in patient pharmacokinetics. With nearly identical drug doses, the homeostatic drug concentrations in different individuals can differ by more than 20-fold, possibly due to differences in absorption, distribution, metabolism, excretion of the drug caused by differences in co-morbid disease, age, concomitant medication, and genetic characteristics of the patients. Different dosage forms of the same drug can cause different concentrations of the drug in vivo due to different absorption degrees and absorption modes. TDM employs quantitative determination of drug concentrations in plasma or serum for dose titration in individual patients in order to obtain optimal therapeutic efficacy, better tolerability, and also to reduce the risk of poisoning. The TDM can also find whether the patient stops taking the medicine, reduces the dosage or takes the medicine excessively in the treatment process in time, help the patient to correctly know the medicine taking, and at present, a considerable amount of psychotropic medicine can adjust the medicine taking dosage according to the plasma concentration of the psychotropic medicine clinically.
The immunoassay-based detection method is the most common TDM method and has the characteristics of accuracy, rapidness and convenience. It adopts the specific reaction of antigen and antibody of the substance to be detected, and utilizes the method of enzyme and substrate or fluorescent label to produce chemical reaction for detection so as to make quantitative determination. However, when clinical drugs to be tested are tested together with other interferents with similar structures, the immunological algorithm has certain limitations. Because the chemical structures of the medicines are similar and the medicines have similar antigen surface regions, antigen-antibody reactions can occur, and the medicines are difficult to be completely distinguished by adopting an immunization method, so that the detected medicine concentration has deviation from the actual condition, and the curative effect and the adverse reaction cannot be well predicted and evaluated.
In recent years, liquid chromatography tandem mass spectrometry (LC-MS/MS) has been widely used in TDM, which has high specificity, short running time, and can simultaneously determine and analyze a variety of chemical structures. In the existing method, for example, a method for determining concentrations of aripiprazole, clozapine, chlorpromazine, risperidone and 9-OH risperidone in human serum disclosed in Chinese patent CN111077239A and a method for detecting risperidone and 9-OH risperidone in plasma disclosed in Chinese patent CN106918675A have the advantages that most of flux is small, only one or more drugs can be determined simultaneously, dozens of drugs are rarely involved, and the detection efficiency is influenced. In the existing methods, for example, "a method and a kit for simultaneously determining 35 psychotropic drugs by high performance liquid chromatography and mass spectrometry" disclosed in chinese patent CN109655568A and "a method for simultaneously detecting 17 antipsychotic drugs in a blood sample" disclosed in chinese patent CN109668979A, the external standard method is used to determine the concentrations of a plurality of drugs, which not only has a matrix effect and cannot be accurately quantified, but also has the problems of too high cost and resource waste due to too many types of the detected drugs, low sensitivity and relatively low sensitivity of a plurality of target substances in the process of simultaneously detecting a plurality of drugs, and influences the detection accuracy, so that a reasonable method needs to be developed in combination with clinical requirements. In addition, some methods cannot detect drugs and active metabolites at the same time, and some drugs not only have pharmacological activity by themselves, but also have the same pharmacological action as the original drugs, for example, clozapine and desmethylclozapine, aripiprazole and dehydroaripiprazole, venlafaxine and O-desmethylvenlafaxine, fluoxetine and norfluoxetine, quetiapine and N-dealkylquetiapine, etc., so that the active metabolites of the drugs are also included in the monitoring range, and the correlation between the blood concentration of a patient and the treatment effect can be better explained.
In summary, the main drawbacks of the prior art clinical testing procedures for psychotropic drug detection are:
there is a matrix effect: part of methods still adopt an external standard method to detect the concentration of the drug, have matrix effect and cannot accurately quantify, most of the existing LC-MS/MS methods adopt isotope substitution and other internal standards to correct the influence of matrix effect and the like, and the external standard method is gradually eliminated.
Detection of drug species not applicable to clinical use: the detection of single or excessive medicine type has the defects of single detection medicine type and low flux, thus influencing the detection efficiency and being not beneficial to developing more detection items; the detection of too many types of medicines causes too high cost and resource waste, and is not easy for large-batch clinical detection.
The linear range is not appropriate: the linear range of various medicines in the measurement method in the prior art is unreasonable, and the method is not suitable for clinical examination without clinical sample verification.
Low sensitivity: in the process of simultaneously detecting a plurality of medicines, the problem of relatively low sensitivity of a plurality of target objects exists, and the detection accuracy is influenced.
Some methods fail to detect both drugs and active metabolites simultaneously: some medicines not only have pharmacological activity, but also have the same pharmacological action as the original medicine, so that the active metabolites of the medicines are also brought into the monitoring range, and the correlation between the blood concentration and the treatment effect of a patient can be better explained.
Disclosure of Invention
Based on the problems in the prior art, the invention provides a detection method for measuring 23 psychotropic drugs and main metabolites in a human serum sample, which has the characteristics of simple and convenient sample pretreatment, small required sample volume, high sensitivity, high accuracy and high flux.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a detection method for determining 23 psychotropic drugs and metabolites in human serum comprises the steps of carrying out sample pretreatment on a human serum sample to be detected, respectively carrying out liquid chromatography tandem mass spectrometry detection on each pretreated sample, and simultaneously accurately and quantitatively analyzing the 23 psychotropic drugs and the metabolites in the human serum; the psychotropic drugs and metabolites include one or more of risperidone, 9-hydroxyrisperidone, olanzapine, quetiapine, N-dealkylquetiapine, aripiprazole, dehydroaripiprazole, amisulpride, clozapine, N-desmethylclozapine, ziprasidone, venlafaxine, O-desmethylvenlafaxine, levetiracetam, lamotrigine, oxcarbazepine, 10-hydroxycarbamazepine, fluoxetine, norfluoxetine, mirtazapine, escitalopram, sertraline, duloxetine; in the liquid chromatography tandem mass spectrometry detection, mass spectrometry adopts multi-reaction monitoring, and the MRM parameters of the compounds are as follows: :
and (3) adopting ESI ion source and positive ion mode segmented scanning analysis.
According to the scheme, the detection method for detecting 23 psychotropic drugs and metabolites in human serum adopts an internal standard method, and 9 stable isotope internal standards are used, namely quetiapine-d8, risperidone-d4, olanzapine-d8, aripiprazole-d8, amisulpride-d5, ziprasidone-d8, fluoxetine-d6, citalopram-d4 and levetiracetam-d 6.
According to the scheme, the detection method for determining the 23 psychotropic drugs and the metabolites in the human serum comprises the following detailed steps:
step S1, preparing a calibrator solution, a quality control solution and an internal standard working solution with series concentrations;
step S2, adding internal standard working solution into the sample to be detected, and carrying out vortex mixing;
and step S3, centrifuging each sample mixed in the step S2, adding diluent into the supernatant after centrifugation, diluting, and performing LC-MS/MS analysis.
According to the scheme, in the step S2, the internal standard working solution is methanol or acetonitrile solution containing an internal standard, and is added according to the addition amount which is 2-5 times of the volume of the sample to be detected; the diluent in the step S3 is 0.1% formic acid aqueous solution, and is added according to the volume of 10-30 times.
According to the above scheme, the conditions of the liquid chromatography are as follows:
and (3) analyzing the column: agilent Eclipse Plus C18 RRHD 1.8 μm 2.1X 50 mm;
mobile phase: phase A: an aqueous solution containing 0.1% formic acid; phase B: acetonitrile solution containing 0.1% formic acid;
elution gradient: mobile phase A + mobile phase B is 100%; keeping the volume of the mobile phase A at 95% for 0-0.8 min; the volume of the mobile phase A is reduced from 95% to 80% in 0.8-2.0 min; 2.0-7.0 min, the volume of the mobile phase A is reduced from 80% to 62%; 7.0-8.0 min, the volume of the mobile phase A is reduced from 62% to 5%; 8.0-9.0 min, keeping the volume of the mobile phase A at 5%; 9.0-9.01 min, increasing the volume of the mobile phase A from 5% to 95%; 9.01-10.0 min, keeping the volume of the mobile phase A at 95%;
flow rate: 0.35 mL/min; column temperature: at 40 ℃.
According to the above scheme, the mass spectrum conditions are as follows:
ion source temperature (tem)): at 450 ℃; ion Source atomizing Gas (Ion Source Gas1(GS 1)): 35 psi; the Ion Source heats the auxiliary Gas (Ion Source Gas2(GS 2)): 35 psi; air Curtain Gas (CUR)): 20 psi; spray capillary Voltage (ion spray Voltage (IS)): 5500V; collision Gas (CAD)): 9.
the invention has the beneficial effects that: by combining clinical detection requirements, a liquid chromatography-tandem mass spectrometry detection method for simultaneously detecting 23 commonly used psychotropic drugs and metabolites thereof is provided, and stable isotope internal standards (9 types) are used to simultaneously and accurately quantify the 23 psychotropic drugs and the metabolites thereof. Respectively selecting a pair of quantitative ion pairs for each substance to be detected, taking the corresponding retention time as a qualitative basis, and making a standard curve for quantification by using a standard substance; and the accuracy and the effectiveness of the quality control product inspection method at low, medium and high levels are applied; meanwhile, the stable isotope internal standard is used for correction, so that the matrix effect can be effectively avoided.
Drawings
FIG. 1 is a standard curve diagram and a representative chromatogram of quetiapine determined by the method of the present invention (the stable isotope internal standard is quetiapine-d 8);
FIG. 2 is a standard curve diagram and a representative chromatogram of the determination of N-dealkylquetiapine by the method of the present invention (the employed stable isotope internal standard is ziprasidone-d 8);
FIG. 3 is a standard curve diagram and a representative chromatogram of risperidone measurement according to the present invention (risperidone-d4 is used as an internal stable isotope);
FIG. 4 is a standard curve diagram and a representative chromatogram of the method of the present invention for measuring 9-hydroxyrisperidone (the stable isotope internal standard is risperidone-d 4);
FIG. 5 is a standard graph and a representative chromatogram of olanzapine measured by the method of the present invention (the employed stable isotope internal standard is olanzapine-d 8);
FIG. 6 is a standard curve diagram and a representative chromatogram of aripiprazole measured by the method of the present invention (the employed stable isotope internal standard is aripiprazole-d 8);
FIG. 7 is a standard curve diagram and a representative chromatogram of the dehydroaripiprazole assay of the present invention (the employed stable isotope internal standard is aripiprazole-d 8);
FIG. 8 is a standard curve diagram and a representative chromatogram of amisulpride measured by the method of the present invention (the adopted stable isotope internal standard is amisulpride-d 5);
FIG. 9 is a standard curve diagram and representative chromatogram of ziprasidone assay using the method of the present invention (ziprasidone-d8 is the internal standard of stable isotope);
FIG. 10 is a standard curve diagram and a representative chromatogram of fluoxetine measurement by the method of the present invention (the stable isotope internal standard is fluoxetine-d 6);
FIG. 11 is a standard curve and representative chromatogram of norfluoxetine assay of the present invention (stable isotope internal standard: fluoxetine-d 6);
FIG. 12 is a standard curve and representative chromatogram of sertraline assay according to the present invention (internal stable isotope is fluoxetine-d 6);
FIG. 13 is a standard curve and a representative chromatogram of duloxetine measurements according to the present invention (stable isotope internal standard: fluoxetine-d 6);
FIG. 14 is a standard graph and a representative chromatogram of mirtazapine measured by the method of the present invention (using a stable isotope internal standard of fluoxetine-d 6);
FIG. 15 is a standard curve diagram and a representative chromatogram of venlafaxine measured by the method of the present invention (the employed stable isotope internal standard is levetiracetam-d 6);
FIG. 16 is a standard curve diagram and a representative chromatogram of the O-desmethylvenlafaxine assay of the present invention (levetiracetam-d6 as an internal stable isotope standard);
FIG. 17 is a standard curve diagram and a representative chromatogram of oxcarbazepine measured by the method of the present invention (the stable isotope internal standard is levetiracetam-d 6);
FIG. 18 is a standard curve diagram and a representative chromatogram of the method of the present invention for the determination of 10-hydroxycarbazepine (the employed stable isotope internal standard is levetiracetam-d 6);
FIG. 19 is a standard curve diagram and a representative chromatogram of levetiracetam determination by the method of the present invention (levetiracetam-d6 is used as an internal stable isotope standard);
FIG. 20 is a standard curve diagram and a representative chromatogram of lamotrigine assay according to the present invention (levetiracetam-d6 is used as an internal standard of stable isotope);
FIG. 21 is a standard curve diagram and a representative chromatogram of clozapine measured by the method of the present invention (the employed stable isotope internal standard is levetiracetam-d 6);
FIG. 22 is a standard curve and representative chromatogram for N-desmethylclozapine measurement according to the present invention (using as internal stable isotope label levetiracetam-d 6);
FIG. 23 is a standard curve and representative chromatogram of escitalopram measured by the method of the present invention (citalopram-d4 as an internal stable isotope);
FIG. 24 is a total chromatogram of 23 mixed standards of psychotropic drugs measured by the method of the present invention.
The attached drawings are LC-MS/MS detection analysis result graphs which are result displays in the embodiment, characters in the graphs are result displays, and the results change according to the result of each detection analysis, namely the characters in the graphs are irrelevant to whether the detection method provided by the invention can be repeatedly implemented, and the characters in the graphs are unclear, so that a person skilled in the art can repeatedly implement the detection method provided by the invention.
Detailed Description
The technical solution of the present invention will be described below with reference to the specific embodiments and the accompanying drawings.
A detection method for determining 23 psychotropic drugs and metabolites in human serum comprises the steps of carrying out sample pretreatment on a human serum sample to be detected, wherein 9 stable isotope internal standard substances are used, namely quetiapine-d8, risperidone-d4, olanzapine-d8, aripiprazole-d8, amisulpride-d5, ziprasidone-d8, fluoxetine-d6, citalopram-d4 and levetiracetam-d6, carrying out liquid chromatography tandem mass spectrometry detection on each pretreated sample, and simultaneously accurately and quantitatively analyzing 23 psychotropic drugs and metabolites in human serum, wherein the psychotropic drugs and metabolites comprise risperidone, 9-hydroxypiperidone, olanzapine, quetiapine, N-dealkylquetiapine, aripiprazole, dehydroaripiprazole, sulpiride, clozapine, N-desmethylclozapine, Ziprasidone, venlafaxine, O-desmethylvenlafaxine, levetiracetam, lamotrigine, oxcarbazepine, 10-hydroxycarbamazepine, fluoxetine, norfluoxetine, mirtazapine, escitalopram, sertraline, duloxetine, comprising the following detailed steps:
step S1, preparing a calibrator solution, a quality control solution and an internal standard working solution with series concentrations;
step S11 preparation of stable isotope internal standard stock solution: appropriate amounts of standard Quetiapine-D8 (Quetiapine-D8Fumarate), Risperidone-D4 (Risperidone-D4), Olanzapine-D8 (Olanzapine-D8), Aripiprazole-D8 (Aripiprazole-D8), Amisulpride-D5 (Amisulpride-D5), Ziprasidone-D8 (Ziprasidone-D8), Fluoxetine-D6 (Fluoxetine-D6 oxalate), Citalopram-D4 (Citalopram-D4), Levetiracetam-D6 (Levetiracetam-D6) were accurately weighed, and stable internal standard solutions of isotopes at a concentration of 1mg/mL were prepared using organic solvents such as methanol, dimethyl sulfoxide (DMSO), etc., respectively.
Step S12 preparation of stable isotope internal standard intermediate working solution: and (4) accurately transferring the stable isotope internal standard stock solution prepared in the step S11, and using methanol as diluent to respectively dilute into internal standard intermediate working solution with the mass concentration of 200 mug/mL.
Step S13 preparation of stable isotope internal standard working solution: accurately transferring the stable isotope internal standard intermediate working solution prepared in the step S12, uniformly mixing, and preparing to obtain a stable isotope internal standard working solution by using acetonitrile as a diluent; the stable isotope internal standard working solution simultaneously comprises: the concentration of quetiapine-d8 is 150ng/mL, the concentration of risperidone-d4 is 10ng/mL, the concentration of olanzapine-d8 is 10ng/mL, the concentration of aripiprazole-d8 is 75ng/mL, the concentration of amisulpride-d5 is 50ng/mL, the concentration of ziprasidone-d8 is 50ng/mL, the concentration of fluoxetine-d6 is 150ng/mL, the concentration of citalopram-d4 is 18ng/mL, and the concentration of levetiracetam-d6 is 100 ng/mL.
Step S14 preparation of standard stock solution: accurately weighing appropriate amounts of lamotrigine, oxcarbazepine, 10-hydroxycarbamazepine, levetiracetam, quetiapine, N-dealkylquetiapine, risperidone, 9-hydroxypiperidone, olanzapine, aripiprazole, dehydroaripiprazole, clozapine, N-desmethylclozapine, amisulpride, ziprasidone, fluoxetine, norfluoxetine, sertraline, escitalopram, duloxetine, mirtazapine, venlafaxine, and O-desmethylvenlafaxine standards, respectively, dissolving in methanol and formulating into standard stock solutions at concentrations as given in the following table:
step S15 preparation of intermediate working solution of mixed standard product: accurately transferring the stock solution of each standard substance prepared in the step S14 in sequence, and preparing an intermediate working solution of the mixed standard substance by using methanol as a diluent, wherein the concentration is shown in the following table;
| name of Compound | Concentration (μ g/mL) | Name of Compound | Concentration (μ g/mL) |
| |
0 | Clozapine | 108.0 |
| Oxcarbazepine | 0 | N-desmethylclozapine | 108.0 |
| 10- |
0 | Amisulpride | 64.8 |
| |
0 | Ziprasidone derivatives | 43.2 |
| Quetiapine | 81 | Fluoxetine | 108.0 |
| N- |
54 | Norfluoxetine | 80.0 |
| Risperidone | 16.2 | Sertraline | 32.4 |
| 9-Hydroxyrisperidone | 16.2 | Escitalopram | 21.6 |
| Olanzapine | 16.2 | Duloxetine | 27.0 |
| Aripiprazole | 108.0 | Mirtazapine | 10.8 |
| Dehydroaripiprazole | 108.0 | Venlafaxine | 81.0 |
| O-desmethylvenlafaxine | 81.0 |
Step S16 preparation of mixed standard working solution: accurately transferring the lamotrigine standard substance stock solution prepared in the step S14, the oxcarbazepine standard substance stock solution, the 10-hydroxycarbazepine standard substance stock solution, the levetiracetam standard substance stock solution and the mixed standard substance intermediate working solution prepared in the step S15 in sequence, and using human serum as diluent to respectively prepare 7 gradient mixed standard substance working solutions, wherein the concentration is shown in the following table;
| serial number | Name of Compound | C1 | C2 | C3 | C4 | | C6 | C7 | |
| 1 | |
18 | 37.5 | 75 | 225 | 360 | 450 | 900 | |
| 2 | N- |
12 | 25 | 50 | 150 | 240 | 300 | 600 | |
| 3 | Risperidone | 3.6 | 7.5 | 15 | 45 | 72 | 90 | 180 | |
| 4 | 9-Hydroxyrisperidone | 3.6 | 7.5 | 15 | 45 | 72 | 90 | 180 | |
| 5 | Olanzapine | 3.6 | 7.5 | 15 | 45 | 72 | 90 | 180 | |
| 6 | |
24 | 50 | 100 | 300 | 480 | 600 | 1200 | |
| 7 | |
24 | 50 | 100 | 300 | 480 | 600 | 1200 | |
| 8 | |
24 | 50 | 100 | 300 | 480 | 600 | 1200 | |
| 9 | N- |
24 | 50 | 100 | 300 | 480 | 600 | 1200 | |
| 10 | Amisulpride | 14.4 | 30 | 60 | 180 | 288 | 360 | 720 | |
| 11 | Ziprasidone derivatives | 9.6 | 20 | 40 | 120 | 192 | 240 | 480 | |
| 12 | |
24 | 50 | 100 | 300 | 480 | 600 | 1200 | |
| 13 | Norfluoxetine | 17.8 | 37.0 | 74.0 | 222.0 | 355.6 | 444.0 | 888.0 | |
| 14 | Sertraline | 7.2 | 15 | 30 | 90 | 144 | 180 | 360 | |
| 15 | Escitalopram | 4.8 | 10 | 20 | 60 | 96 | 120 | 240 | |
| 16 | Duloxetine | 6 | 12.5 | 25 | 75 | 120 | 150 | 300 | |
| 17 | |
18 | 37.5 | 75 | 225 | 360 | 450 | 900 | |
| 18 | O- |
18 | 37.5 | 75 | 225 | 360 | 450 | 900 | |
| 19 | Mirtazapine | 2.4 | 5 | 10 | 30 | 48 | 60 | 120 | |
| 20 | Oxcarbazepine | 1.2 | 2.5 | 5 | 15 | 24 | 30 | 60 | |
| 21 | 10-hydroxy carbamazepine | 1.2 | 2.5 | 5 | 15 | 24 | 30 | 60 | |
| 22 | Levetiracetam | 1.2 | 2.5 | 5 | 15 | 24 | 30 | 60 | |
| 23 | Lamotrigine | 0.3 | 0.625 | 1.25 | 3.75 | 6 | 7.5 | 15 |
In the above table, the concentration units of oxcarbazepine, 10-hydroxycarbamimepine, levetiracetam and lamotrigine are all μ g/mL, and the rest are all ng/mL;
step S17 preparation of mixed quality control working solution: accurately transferring the lamotrigine standard substance stock solution prepared in the step S14, the oxcarbazepine standard substance stock solution, the 10-hydroxycarbazepine standard substance stock solution, the levetiracetam standard substance stock solution and the mixed standard substance intermediate working solution prepared in the step S15 in sequence, and using human serum as diluent to respectively prepare mixed quality control working solutions with low, medium and high concentration levels, wherein the concentrations are shown in the following table;
| serial number | Name of Compound | | MQC | HQC | |
| 1 | |
54 | 360 | 450 | |
| 2 | N-dealkylated quetiapine | 36 | 240 | 480 | |
| 3 | Risperidone | 10.8 | 72 | 144 | |
| 4 | 9-Hydroxyrisperidone | 10.8 | 72 | 144 | |
| 5 | Olanzapine | 10.8 | 72 | 144 | |
| 6 | |
72 | 480 | 960 | |
| 7 | |
72 | 480 | 960 | |
| 8 | |
72 | 480 | 960 | |
| 9 | N- |
72 | 480 | 960 | |
| 10 | Amisulpride | 43.2 | 288 | 576 | |
| 11 | Ziprasidone derivatives | 28.8 | 192 | 384 | |
| 12 | |
72 | 480 | 960 | |
| 13 | Norfluoxetine | 53.3 | 355.6 | 711.1 | |
| 14 | Sertraline | 21.6 | 144 | 288 | |
| 15 | Escitalopram | 14.4 | 96 | 192 | |
| 16 | |
18 | 120 | 240 | |
| 17 | |
54 | 360 | 720 | |
| 18 | O- |
54 | 360 | 720 | |
| 19 | Mirtazapine | 7.2 | 48 | 96 | |
| 20 | Oxcarbazepine | 3.6 | 24 | 48 | |
| 21 | 10-hydroxy carbamazepine | 3.6 | 24 | 48 | |
| 22 | Levetiracetam | 3.6 | 24 | 48 | |
| 23 | Lamotrigine | 0.9 | 6 | 12 |
In the above table, the concentration units of oxcarbazepine, 10-hydroxycarbamimepine, levetiracetam and lamotrigine are all μ g/mL, and the rest are all ng/mL;
step S2, adding the sample to be measured into a sample container according to the volume ratio of 1: 4, adding internal standard working solution with 4 times volume amount, and mixing in a vortex manner;
step S3, centrifuging each sample mixed in the step S2, and taking supernatant according to a volume ratio of 1: 20 was diluted with a 20-fold volume of 0.1% formic acid in water and analyzed by LC-MS/MS.
The conditions of the liquid chromatography are as follows:
and (3) analyzing the column: agilent Eclipse Plus C18 RRHD 1.8 μm 2.1X 50 mm;
mobile phase: phase A: an aqueous solution containing 0.1% formic acid; phase B: acetonitrile solution containing 0.1% formic acid;
elution gradient: mobile phase A + mobile phase B is 100%; keeping the volume of the mobile phase A at 95% for 0-0.8 min; the volume of the mobile phase A is reduced from 95% to 80% in 0.8-2.0 min; 2.0-7.0 min, the volume of the mobile phase A is reduced from 80% to 62%; 7.0-8.0 min, the volume of the mobile phase A is reduced from 62% to 5%; 8.0-9.0 min, keeping the volume of the mobile phase A at 5%; 9.0-9.01 min, increasing the volume of the mobile phase A from 5% to 95%; 9.01-10.0 min, keeping the volume of the mobile phase A at 95%;
flow rate: 0.35 mL/min; column temperature: at 40 ℃.
The mass spectrum conditions were as follows:
ion source temperature (tem)): at 450 ℃; ion Source atomizing Gas (Ion Source Gas1(GS 1)): 35 psi; the Ion Source heats the auxiliary Gas (Ion Source Gas2(GS 2)): 35 psi; air Curtain Gas (CUR)): 20 psi; spray capillary Voltage (ion spray Voltage (IS)): 5500V; collision Gas (CAD)): 9; mass spectra were monitored using multiple reactions, and compound MRM parameters were as follows:
and (3) adopting ESI ion source and positive ion mode segmented scanning analysis.
Detecting the mixed standard working solution with the 7 gradients by using a high performance liquid chromatography-mass spectrometer to obtain chromatographic peak areas of 7 standard solutions with different concentrations, respectively taking the ratio of the chromatographic peak areas of the 7 standard solutions with different concentrations to the chromatographic peak area of a corresponding isotope internal standard as a vertical coordinate y of a standard curve equation, taking the concentrations of the 7 standard solutions with different concentrations as horizontal coordinates x of a standard curve, performing linear regression on the detected data with 7 different concentrations, and fitting to obtain standard curve equations corresponding to all the standards: and y is a x + b, and the result is shown in the figure.
The detection method provided by the invention is further subjected to feasibility detection, and the process and the result are as follows:
firstly, the method provided by the invention is further subjected to the precision analysis in batch and between batches, and the process and the result are as follows:
precision describes how closely an analyte is determined repeatedly, and the relative standard deviation (coefficient of variation, CV%) defined as the measured value should be obtained using the results of analyzing batch samples with the same proof of accuracy as that of the proof of accuracy, to obtain precision of quality control samples of low, medium and high concentrations, both within the same batch (intra-batch precision) and between different batches (inter-batch precision).
Internal precision: the invention adopts blank human serum to prepare low, medium and high concentration mixed quality control working solution; each concentration was tested in 6 replicates and assayed for 1 day.
Batch precision: the invention adopts blank human serum to prepare low, medium and high concentration mixed quality control working solution; each concentration was tested in 6 replicates for 3 consecutive days.
The result of the batch precision detection is as follows: the inter-batch variation coefficient (CV%) of 23 analytes to be detected is less than or equal to 15 percent; the detection result of the internal precision of the batch is as follows: the intra-batch variation coefficient (CV%) of all analytes to be detected is less than or equal to 15%.
And (3) residual test:
the residue should be estimated by injecting a blank sample after injecting the high concentration sample or the calibration standard to the extent that the residue in the blank sample after injecting the residual high concentration sample should not exceed 20% of the lower limit of quantitation.
The invention adopts blank human serum to prepare a calibration standard sample ULOQ, and the calibration standard sample ULOQ is evaluated by continuously injecting 5 needles of blank samples after the calibration standard sample ULOQ is injected; the measurement is carried out for 3 days continuously, and the detection result shows that: the residues in blank samples after ULOQ for 23 analytes to be tested were all less than 20% LLOQ;
and (3) testing matrix effect:
the present invention adopts 6 batches of blank human serum matrices from different donors to investigate the matrix effect of analytes to be tested under the method at low and high concentrations.
For each batch of matrix, calculating the ratio of the peak area (measured by adding a mixed standard working solution and a stable isotope internal standard after extracting blank human serum matrix) in the presence of the matrix to the corresponding peak area (pure solution of the analyte and the stable isotope internal standard) of the matrix without human serum to calculate the matrix factor of each analyte and the stable isotope internal standard; and further dividing the matrix factor of the analyte by the matrix factor of the corresponding stable isotope internal standard to calculate the matrix factor normalized by the internal standard, wherein the detection results are shown in the following table, and the variation coefficients of the matrix factors normalized by the stable isotope internal standard calculated by 23 analytes to be detected from 6 batches of matrixes are all less than 15%, which indicates that the method has no obvious matrix effect.
The present invention is provided by the above embodiments only for illustrating and not limiting the technical solutions of the present invention, and although the above embodiments describe the present invention in detail, those skilled in the art should understand that: modifications and equivalents may be made thereto without departing from the spirit and scope of the invention and any modifications and equivalents may fall within the scope of the claims.
Claims (6)
1. A detection method for determining 23 psychotropic drugs and metabolites in human serum is characterized in that a human serum sample to be detected is subjected to sample pretreatment, liquid chromatography tandem mass spectrometry detection is respectively carried out on each pretreated sample, and the 23 psychotropic drugs and the metabolites in the human serum are accurately and quantitatively analyzed; the psychotropic drugs and metabolites include one or more of risperidone, 9-hydroxyrisperidone, olanzapine, quetiapine, N-dealkylquetiapine, aripiprazole, dehydroaripiprazole, amisulpride, clozapine, N-desmethylclozapine, ziprasidone, venlafaxine, O-desmethylvenlafaxine, levetiracetam, lamotrigine, oxcarbazepine, 10-hydroxycarbamazepine, fluoxetine, norfluoxetine, mirtazapine, escitalopram, sertraline, duloxetine; in the liquid chromatography tandem mass spectrometry detection, mass spectrometry adopts multi-reaction monitoring, and the MRM parameters of the compounds are as follows:
and (3) adopting ESI ion source and positive ion mode segmented scanning analysis.
2. The detection method for detecting 23 psychotropic drugs and metabolites in human serum according to claim 1, wherein the internal standard method is adopted, and 9 internal standard substances with stable isotopes are used, namely quetiapine-d8, risperidone-d4, olanzapine-d8, aripiprazole-d8, amisulpride-d5, ziprasidone-d8, fluoxetine-d6, citalopram-d4 and levetiracetam-d 6.
3. The method for detecting 23 psychotropic drugs and metabolites in human serum according to claim 1, wherein the method comprises the following detailed steps:
step S1, preparing a calibrator solution, a quality control solution and an internal standard working solution with series concentrations;
step S2, adding internal standard working solution into the sample to be detected, and carrying out vortex mixing;
and step S3, centrifuging each sample mixed in the step S2, adding diluent into the supernatant after centrifugation, diluting, and performing LC-MS/MS analysis.
4. The method for detecting 23 psychotropic drugs and metabolites in human serum according to claim 3, wherein in step S2, the internal standard working solution is a methanol or acetonitrile solution containing an internal standard, and is added in an amount of 2-5 times the volume of the sample to be detected; the diluent in the step S3 is 0.1% formic acid aqueous solution, and is added according to the volume of 10-30 times.
5. The method for detecting 23 psychotropic drugs and metabolites in human serum according to claim 3, wherein the conditions of the liquid chromatography are as follows:
and (3) analyzing the column: agilent Eclipse Plus C18 RRHD 1.8 μm 2.1X 50 mm;
mobile phase: phase A: an aqueous solution containing 0.1% formic acid; phase B: acetonitrile solution containing 0.1% formic acid;
elution gradient: mobile phase A + mobile phase B is 100%; keeping the volume of the mobile phase A at 95% for 0-0.8 min; the volume of the mobile phase A is reduced from 95% to 80% in 0.8-2.0 min; 2.0-7.0 min, the volume of the mobile phase A is reduced from 80% to 62%; 7.0-8.0 min, the volume of the mobile phase A is reduced from 62% to 5%; 8.0-9.0 min, keeping the volume of the mobile phase A at 5%; 9.0-9.01 min, increasing the volume of the mobile phase A from 5% to 95%; 9.01-10.0 min, keeping the volume of the mobile phase A at 95%;
flow rate: 0.35 mL/min; column temperature: at 40 ℃.
6. The method for detecting 23 psychotropic drugs and metabolites in human serum according to claim 5, wherein the mass spectrometric conditions are as follows:
ion source temperature: at 450 ℃; ion source atomization gas: 35 psi; heating auxiliary gas by an ion source: 35 psi; air curtain air: 20 psi; spray capillary voltage: 5500V; collision gas: 9.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114755348A (en) * | 2022-06-13 | 2022-07-15 | 北京和合医学诊断技术股份有限公司 | Method for simultaneously detecting contents of 20 medicines and metabolites thereof |
| CN114935620A (en) * | 2022-05-20 | 2022-08-23 | 上海市精神卫生中心（上海市心理咨询培训中心） | Kit for simultaneously and quantitatively detecting 78 neuropsychiatric drugs |
| CN114994198A (en) * | 2022-05-20 | 2022-09-02 | 上海市精神卫生中心（上海市心理咨询培训中心） | Method for simultaneously and quantitatively detecting 78 neuropsychiatric drugs by liquid chromatography-mass spectrometry |
| CN115290775A (en) * | 2022-07-25 | 2022-11-04 | 上海市徐汇区中心医院 | Quality control product of mental drugs, kit, preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170138940A1 (en) * | 2014-04-28 | 2017-05-18 | Magnamedics Gmbh | Method for isolating trace components from a biological liquid sample |
| CN109655568A (en) * | 2019-01-22 | 2019-04-19 | 杭州度安医学检验实验室有限公司 | Efficient LC-MS measures the method and kit of 35 kinds of psychotropic agents simultaneously |
| CN110531014A (en) * | 2019-03-11 | 2019-12-03 | 成都民用航空医学中心 | The method that Liquid Chromatography-Tandem Mass Spectrometry detects 43 kinds of drugs in blood |
-
2021
- 2021-10-09 CN CN202111174772.3A patent/CN113933423B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170138940A1 (en) * | 2014-04-28 | 2017-05-18 | Magnamedics Gmbh | Method for isolating trace components from a biological liquid sample |
| CN109655568A (en) * | 2019-01-22 | 2019-04-19 | 杭州度安医学检验实验室有限公司 | Efficient LC-MS measures the method and kit of 35 kinds of psychotropic agents simultaneously |
| CN110531014A (en) * | 2019-03-11 | 2019-12-03 | 成都民用航空医学中心 | The method that Liquid Chromatography-Tandem Mass Spectrometry detects 43 kinds of drugs in blood |
Non-Patent Citations (3)
| Title |
|---|
| PAULA PROENÇA等: "Identification and Quantification of Antipsychotics in Blood Samples by LC–MS-MS: Case Reports and Data from Three Years of Routine Analysis", 《JOURNAL OF ANALYTICAL TOXICOLOGY》 * |
| 侯峰等: "毛发中8种常见精神类药物的LC-MS/MS检测方法", 《中国药物依赖性杂志》 * |
| 张秀尧等: "超高效液相色谱-串联质谱法同时快速确证检测血浆和 尿液中的42种精神药物及其代谢产物", 《色谱》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114935620A (en) * | 2022-05-20 | 2022-08-23 | 上海市精神卫生中心（上海市心理咨询培训中心） | Kit for simultaneously and quantitatively detecting 78 neuropsychiatric drugs |
| CN114994198A (en) * | 2022-05-20 | 2022-09-02 | 上海市精神卫生中心（上海市心理咨询培训中心） | Method for simultaneously and quantitatively detecting 78 neuropsychiatric drugs by liquid chromatography-mass spectrometry |
| CN114755348A (en) * | 2022-06-13 | 2022-07-15 | 北京和合医学诊断技术股份有限公司 | Method for simultaneously detecting contents of 20 medicines and metabolites thereof |
| CN114755348B (en) * | 2022-06-13 | 2022-09-20 | 北京和合医学诊断技术股份有限公司 | Method for simultaneously detecting contents of 20 medicines and metabolites thereof |
| CN115290775A (en) * | 2022-07-25 | 2022-11-04 | 上海市徐汇区中心医院 | Quality control product of mental drugs, kit, preparation method and application thereof |
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